With some degree of irony, acceptance of the hypothesis that the innate immune system positively stimulates the development of asthma came with the understanding that airway epithelial cells might also be a source of Th2 cytokines. In particular, evidence arose that epithelial production of IL-25, thymic stromal lymphopoietin (TSLP), and/or IL-33 might drive Th2 responses (particularly in the form of IL-13 production) and in turn result in disease traits that are characteristic of asthma (73
). This issue naturally led to the search for innate immune cells that might respond to epithelial cytokines with an increase in IL-13 production. A series of reports identified lineage-negative lymphoid cells (analogous to those found in the gut) that responded to IL-25 and IL-33 by producing IL-13 and IL-5 (85
). Follow-up reports noted that these relatively rare cells (variously named non-B/non-T cells, natural helper cells, nuocytes, or innate lymphoid cells) constitute the major source of IL-13 in the lung after challenge with an allergen (ovalbumin) or the helminth N. brasiliensis
). This finding is surprising given the usual abundance of eosinophils in these conditions and the high-level capability of eosinophils for IL-13 production. A related report proposed that innate lymphoid cells also mediate the airway inflammation and hyperreactivity that occur just one day after IAV infection in mice (91
). In this case, the virus activates alveolar macrophages to generate IL-33 that stimulates innate lymphoid cells to produce IL-13. However, we are still missing an experiment that selectively blocks endogenous innate lymphoid cells and demonstrates the contribution of the native cell population to the asthma phenotype. This step will be critical to use these immune cell events as biomarkers and therapeutic targets in humans.
Another noteworthy drawback to studies of asthma is the frequent lack of physiological relevance of experimental models and the attendant requirement to translate findings in the model to humans with asthma. For example, an obvious experiment in an animal model would be to explore the possible connection between severe RSV bronchiolitis and asthma. However, a human pathogen such as RSV exhibits only limited replication in a rodent host (92
), perhaps explaining the relatively short-lived effect of RSV on airway inflammation and dysfunction in mice. Such an outcome does not fit with the full spectrum of clinical experience in humans, in which the effect of RSV on airway inflammation and the development of postviral asthma might be delayed or might last for weeks, months, or even years. Similarly, despite genetic manipulation to favor infection, HRV also exhibits a relatively low level of replication and consequent illness in the mouse model (93
). Perhaps this experimental difficulty will improve with the isolation of a more aggressive group of HRV-C species (95
). Nonetheless, HRV as well as RSV and IAV infection have all been demonstrated to worsen allergen-induced asthma in experimental models (93
), raising the possibility that virus-allergen synergy is an initiator or perpetuator of disease in childhood and adulthood.
Meanwhile, there was still a need to develop a model of viral infection that resulted in long-term experimental asthma. Toward that end, it is possible to remove the obstacle in host range for RSV (and other human pathogens) by substituting the corresponding mouse paramyxovirus known as mouse parainfluenza virus type I or Sendai virus (SeV). This alternative provides for a model that mimics cardinal features of the human disease process, including acute bronchiolitis (as found in RSV-infected infants) followed by chronic (perhaps lifelong) airway inflammation, mucus overproduction, and hyperreactivity that depend on genetic susceptibility (as found in asthmatic children, teenagers, and adults) (99
). The SeV mouse model has proven useful for understanding immunological events leading to acute illness and chronic airway disease after viral infection (Figure ). In terms of acute postviral illness, the model has allowed for more complete definition of the innate and adaptive immune response that controls and then clears infectious respiratory virus. As introduced above, there is an emerging understanding of precisely how airway epithelial cells participate in this aspect of host defense. In particular, the capacity of airway epithelial cells for IFN production and signaling is emerging as a key requirement for containing viral infection and preventing postviral asthma (46
). In concert with the epithelial contribution for viral control, there is also a better awareness of the role of the lung macrophage in the antiviral host response. For example, an unexpected action of the chemokine CCL5 allows the macrophage to avoid virus-induced cell death and thereby continue the task of viral clearance (103
). We also understand at least some of the pieces for how this innate response cues the subsequent adaptive response to achieve full clearance of the virus and protection again similar viruses in the future. Critical elements include DC arming of CD8+
) T cell cytotoxicity for lysis of infected cells as well as CD4+
T cell help for B cell and plasma cell antiviral antibody production (104
Immune pathways leading to postviral lung disease.
From the perspective of asthma, however, the real advantage of the SeV mouse model is that it manifests the pattern of airway inflammation, hyperreactivity, and mucus overproduction that develop and persist long after clearance of infectious virus. The extended time course of this postviral process therefore better mimics the clinical experience that links severe RSV bronchiolitis to a long-term wheezing illness in early childhood, and perhaps to the chronic obstructive lung disease that makes up the spectrum of asthma and (as we are learning) COPD in later life as well. The model has revealed two new potentially asthmagenic pathways: one leading from conventional DCs to Th2 cells (107
), and another from APCs (DCs or macrophages) to invariant NKT (iNKT) cells to alternatively activated (M2) monocytes and macrophages (109
). The first pathway is responsible for the early/transient disease that appears at 3 weeks after viral inoculation and features IFN-α/β–dependent expression and IgE-dependent activation of the high-affinity IgE receptor (FcεRI) on DCs and consequent CCL28 production to recruit IL-13–producing CD4+
T cells to the airways (107
). The connection between IFN and DCs requires a CD11b-dependent interaction with a CD49+
subset of PMNs (108
). This pathway thereby contains components of both the innate and adaptive immune systems. The second pathway is responsible for the late/chronic disease that develops fully at 7 weeks after inoculation and is driven instead by an innate immune response that relies on iNKT cells that are programmed to activate macrophages to produce IL-13 (109
). The interaction between iNKT cells and macrophages depends on contact between the semi-invariant Vα14Jα18-TCR on lung iNKT cells and the oligomorphic MHC-like protein CD1d on macrophages as well as NKT cell production of IL-13 that binds to the IL-13 receptor (IL-13R) on the macrophage (109
). This pathway appears to function independent of the adaptive immune system. Each of these pathways provides for new biomarkers and therapeutic targets for clinical application.
A critical next step for this approach (and others like it) is to determine whether similar abnormalities of the innate immune system are also found in asthma and in forms of COPD that may also manifest airway inflammation, mucus overproduction, and hyperreactivity. This translation is just beginning, but it has been shown that the high-affinity IgE receptor is found on human DCs in peripheral blood samples from children as early as one year of age (111
). Analysis of the second immune axis is challenging due to the rarity of NKT cells and the need for specific biomarkers of M2 macrophages in lung tissue. These problems can be overcome in part by using whole-lung explants from patients undergoing transplantation for severe COPD that continue to manifest airway inflammation. A proof-of-concept analysis of a small number of these patients provided preliminary evidence of increased numbers of iNKT cells and IL-13–positive macrophages in the lung in concert with mucus overproduction (109
). Subsequent analysis defined chitinase 1 as a relatively selective marker of M2 macrophage activation and found increased chitinase 1 levels in lung as well as peripheral blood samples in a larger group of COPD patients (112
). Here again, the M2 macrophage abnormality was most prominent in those with severe disease. Recent analysis of 15-lipoxygenase-2 (15–LOX-2) as an additional M2 macrophage marker provided similar Results (113
). The analysis of the NKT cell–M2 macrophage axis in asthma is still under study. An initial investigation found increased numbers of iNKT cell levels in samples from allergic asthma patients at baseline (114
), while others suggest no increase at baseline or an increase mainly with allergen challenge or the presence of severe asthma (115
). No studies yet assess the level of iNKT cell activation in asthma (or COPD), which is likely to be more informative that counting rare cell populations. An initial analysis also revealed preliminary evidence of IL-13–positive macrophages in BAL samples from a small number of patients with severe asthma (109
), but markers of M2 macrophage differentiation that are more stable and expressed at higher levels than cytokines are likely to be more reliable. Increased levels of chitinase 3L1 are found in peripheral blood and in lung macrophages (as well as neutrophils and epithelial cells) as a possible sign of M2 macrophage activation in asthma (118
). Here again, the abnormality appeared most prominent in the most severe forms of asthma. Initial work also suggests that expression levels of 15-LOX isoforms (15–LOX-1 versus 15-LOX-2) may be able to discriminate epithelial versus macrophage activation to establish an M2 pattern of immune activation in asthma and COPD (113
). Further work is needed to fully define the upstream immune axis for M2 macrophage activation in asthma, and in particular to determine the relative or perhaps synergistic roles of the innate and adaptive immune responses to viruses and allergens. Nonetheless, these clinical biomarkers might be developed as tools for patient stratification that could prove useful for directed immune therapy.
An additional next step for these studies is to define the relationship of these new immune pathways to the recent observations for epithelial cytokines (especially IL-25, TSLP, and IL-33) and innate lymphoid cells that might also participate in IL-13 production in asthma (119
). Initial work suggests that these pathways are also selectively activated in the SeV mouse model and in patients with COPD, thereby providing further support for a shared immune mechanism among these conditions (45
). There is also a need for a comprehensive analysis of the role of viral recognition receptors, including the TLRs (such as TLR3, -7, and -8), RNA helicases (such as MDA-5 and RIG-I), and NOD-like receptors (such as NALP3) that appear active in the antiviral response (123
). Previous work suggested that activation of TLR3 (as well as TLR7 and MDA5) generally worsened the acute inflammatory response to other viruses, including Th2-dependent inflammation that develops for a short time after RSV infection in mice (94
). However, as noted above, these reports do not address the effect of activation of these viral sensors on chronic obstructive lung disease and therefore do not make a connection between innate immune cell activation and long-term inflammation. As noted below, this immune puzzle needs to be solved to better understand pathogenesis and identify rational and druggable targets for therapeutics in asthma. At present, however, asthma might best be viewed as a complex immune disorder that develops as a function of both innate and adaptive immune responses that contribute to varying degrees in any individual patient. Furthermore, it is intriguing that a distinct innate immune response to viral infection may be linked to the most severe forms of asthma and COPD, in which therapy is most limited and perhaps of greatest priority.